Peptide nucleic acid probes for analysis of pseudomonas (sensu stricto)

ABSTRACT

Disclosed is a PNA probe that includes a nucleobase sequence suitable for the detection, identification and/or quantitation of  Pseudomonas  (sensu stricto). In one embodiment, the PNA probe is complementary to a target sequence of 23S rRNA or rDNA from all species of the genus  Pseudomonas.  The invention has a wide range of important uses including detecting  Pseudomonas  in a sample of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of U. S.application Ser. No. 10/719,979 as filed on Nov. 21, 2003, whichapplication is a continuation of Provisional Application No. 60/428,554,filed on Nov. 22, 2002. The disclosures of the U.S. Ser. No. 10/719979and 60/428,554 applications are each incorporated by reference.

[0002] The present invention relates to peptide nucleic acid (PNA)probes and methods for the analysis of Pseudomonas (sensu stricto)optionally present in a sample. The invention further relates todiagnostic kits comprising such PNA probes.

BACKGROUND OF THE INVENTION

[0003] Detection, identification and quantitation of specificmicroorganisms is fundamental to many areas of microbiology ranging fromthe detection of pathogens in samples of human origin, to spoilageorganisms or pathogens in food and beverages and environmentalcontaminants in municipal water. There are numerous examples whereantibiotic treatment is instituted before the infectious agent has beenconfirmed, food is released for consumption before the microbiologicaltest results are available, or municipal water is distributed viapipelines to the public while culture-based tests are still incubating.The requirement for rapid and accurate test results is obvious.

[0004] Comparative analysis of ribosomal RNA (rRNA) sequences or genomicDNA sequences corresponding to said rRNA (rDNA) has become a widelyaccepted method for establishing phylogenetic relationships betweenbacterial species (Woese, Microbiol. Rev. 51:221-271 (1987)), andBergey's Manual of systematic bacteriology has been revised based onrRNA or rDNA sequence comparisons. Ribosomal RNA or rDNA sequencedifferences between closely related species enable design of specificprobes for microbial identification and thus enable diagnosticmicrobiology to be based on a single genetic marker rather than a seriesof phenotypic markers as characterizing traditional microbiology (Delonget al., Science 342:1360-1363 (1989)).

[0005] The taxonomy of the genus Pseudomonas has been changed in recentyears, such that many species previously classified as Pseudomonasspecies have been reclassified and now belongs to other genera, such asBurkholderia, Xanthomonas, Aeromonas, Brevundimonas etc. However manycurrent methods, such as Pseudomonas specific growth media, are stillbased on the former taxonomy, such the microorganisms identified asPseudomonas (sensu stricto) in fact may be former Pseudomonas speciesnot longer belonging to the Pseudomonas genus (Pacheco & Sage, Abstract,Annual Meeting of the American Society for Microbiology, Salt Lake City,May 2002). There is therefore a need for novel identification methodsreflecting the revised taxonomy of the genus Pseudomonas.

[0006] Despite its name, Peptide Nucleic Acid (PNA) is neither a peptidenor a nucleic acid, it is not even an acid. PNA is a non-naturallyoccuring polyamid that can hybridize to nucleic acid (DNA and RNA) withsequence specificity (See: U.S. Pat. No. 5,539,082) and Egholm et al.,Nature 365:566-568 (1993)) according to Watson-Crick base paring rules.However, whereas nucleic acids are biological materials that play acentral role in the life of living species as agents of genetictransmission and expression, PNA is a recently developed totallyartificial molecule, conceived in the minds of chemists and made usingsynthetic organic chemistry. PNA also differs structurally from nucleicacid. Although both can employ common nucleobases (A, C, G, T, and U),the backbones of these molecules are structurally diverse. The backbonesof RNA and DNA are composed of repeating phosphodiester ribose and2-deoxyribose units. In contrast, the backbones of the most common PNAsare composed on (aminoethyl)-glycine subunits. Additionally, in PNA thenucleobases are connected to the backbone by an additional methylenecarbonyl moiety. PNA is therefore not an acid and therefore contains nocharged acidic groups such as those present in DNA and RNA. Thenon-charged backbone allows PNA probes to hybridize under conditionsthat are destabilizing to DNA and RNA. Attributes that enable PNA probesto access targets, such as highly structured rRNA and double strandedDNA, known to be inaccessible to DNA probes (See: Stephano &Hyidig-Nielsen, IBC Library Series Publication #948. InternationalBusiness Communication, Southborough, Mass., pp.19-37 (1997)). PNAs areuseful candidates for investigation when developing probe-basedhybridization assays because they hybridize to nucleic acids withsequence specificity. However, PNA probes are not the equivalent ofnucleic acid probes in structure or function.

[0007] There is a need in the field for effective PNA probes that can beused to analyze Pseudomonas (sensu stricto) in a wide range of samples.PNA probes targeting Pseudomonas aeruginosa have previously beendescribed (Stender et al., J. Microbiol. Methods 42:245-253 (2000),however the heterogenicity of the species within the genus Pseudomonascomplicates the design of specific PNA probes targeting all species ofthe genus Pseudomonas.

SUMMARY OF THE INVENTION

[0008] This invention is directed to PNA probes and their design as wellas methods and kits useful for analysis of Pseudomonas (sensu stricto)optionally present in a sample of interest. In accordance with claim 1,for instance, the PNA probes are directed to 23S rRNA or the genomicsequences corresponding to said rRNA (rDNA) or its complement.

[0009] These PNA probes have the inherent physico/chemicalcharacteristics of PNA probes as compared to nucleic acid probes, suchthat rapid and accurate analysis can be performed using just a singlePNA probe. Furthermore, PNA probes also offers an advantage as comparedto nucleic acid probes when applied in fluorescence in situhybridization assays. Where nucleic acid probes require fixation andpermeabilization with cross-linking agents and/or enzymes (for examplesee Kempf et al., J. Clin. Microbiol 38:830-838 (2000)), these PNAprobes can be applied directly following smear preparation.

[0010] Accordingly, and in one aspect, the invention features a PNAprobe that includes a nucleobase sequence suitable for the detection,identification and/or quantitation of Pseudomonas (sensu stricto). Inone embodiment, the PNA probe is complementary to a target sequence of23S rRNA or rDNA (or its complement) obtained from essentially anyspecies of the genus Pseudomonas. An important feature of the inventionis that such probes can be used to detect, identify and/or quantitatenearly any species of Pseudomonas as outlined below.

[0011] Such selectivity for Pseudomonas is accomplished through use of asingle probe sequence rather than use of a less specific prior probesand probe sets.

[0012] In a preferred embodiment, these PNA probes have a relative shortnucleobase sequence, such as 15 nucelobases as illustrated in example 1,whereas nucleic acid probes due to their lower Tm values typically haveat least 18 nucleobases (For example see Kempf et al., J. Clin.Microbiol 38:830-838 (2000)). A difference that provides these PNAprobes with better discrimination to closely related non-targetsequences with a single or just a few nucleobase difference(s).

[0013] In another aspect, the invention features a method for thedetection, identification and/or quantitation of Pseudomonas (sensustricto) in a sample. In one embodiment, the method includes: a)contacting at least one of the PNA probes of claims 1-12 to the sample,b) hybridizing the PNA probe to a target sequence of species of thegenus Pseudomonas in the sample; and c) detecting the hybridization asbeing indicative of presence, identity and/or amount of Pseudomonas(sensu stricto) in the sample.

[0014] In one example, the method comprises contacting a sample with aPNA probe having a probing nucleobase sequence of CCT ACC ACC TTA MC(Seq. Id. No. 1) and the complements thereof. According to thisinvention embodiment, the presence, absence and/or number of Pseudomonas(sensu stricto) organisms in the sample are then detected, identifiedand/or quantitated by correlating the hybridization, under suitablehybridization conditions, of the probing nucleobase sequence of theprobe to the target sequence. Consequently, the presence, absence and/ornumber of Pseudomonas (sensu stricto) organisms in the sample aredetermined by direct or indirect detection of the probe/target sequencehybrid.

[0015] In still another embodiment, this invention is directed to kitssuitable for performing an assay that detect, identify and/or quantitatePseudomonas (sensu stricto) optionally present in a sample. The kits ofthis invention comprise one or more PNA probes and other reagents orcompositions that are selected to perform an assay or otherwise simplifythe performance of an assay.

[0016] Thus in one invention embodiment, the kit is suitable to detect,identify and/or quantitate Pseudomonas (sensu stricto) in a sample inwhich the kit includes a) at least one PNA probe as defined herein andb) other reagents or compositions necessary to perform the assay suchas, but not limited to, buffers, stabilizers, water and the like as wellas directions for using the kit.

[0017] Those of ordinary skill in the art will appreciate that asuitable PNA probe need not have exactly these probing nucleobasesequences to be operative but often modified according to the particularassay conditions. For example, shorter PNA probes can be prepared bytruncation of the nucleobase sequence if the stability of the hybridneeds to be modified to thereby lower the Tm and/or adjust forstringency. Similarly, the nucleobase sequence may be truncated by oneend and extended by the other end as long as the discriminatingnucleobases remain within the sequence of the PNA probe. Such variationsof the probing nucleobase sequences within the parameters describedherein are considered to be embodiments of this invention.

[0018] The PNA probe, methods and kits of this invention are bothsensitive and specific for Pseudomonas (sensu stricto). Moreover, theassays described herein are rapid (less than 3 hours) and capable ofanalysis of Pseudomonas (sensu stricto) in a single assay.

[0019] Those of ordinary skill in the art will also appreciate that thecomplement probing sequence is equally suitable for assays, such as butnot limited to real-time PCR, that are using rDNA as target.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS

[0020] a. As used herein, the term “nucleobase” means those naturallyoccurring and those non-naturally occurring heterocyclic moietiescommonly known to those who utilize nucleic acid technology or utilizepeptide nucleic acid technology to thereby generate polymers that cansequence specifically bind to nucleic acids.

[0021] b. As used herein, the term “nucleobase sequence” means anysegment of a polymer that comprises nucleobase-containing subunits.Non-limiting examples of suitable polymers or polymer segments includeoligodeoxynucleotides, oligoribonucleotides, peptide nucleic acids,nucleic acid analogs, nucleic acid mimics, and/or chimeras.

[0022] c. As used herein, the term “target sequence” means thenucleobase sequence that is to be detected in an assay.

[0023] d. As used herein, the term “probe” means a polymer (e. g. a DNA,RNA, PNA, chimera or linked polymer) having a probing nucleobasesequence that is designed to sequence-specifically hybridize to a targetsequence of a target molecule of an organism of interest.

[0024] e. As used herein, “analyzed” means that the individual bacteriaare marked for detection, identification and/or quantitation and/or fordetermination of resistance to antibiotics (antimicrobialsusceptibility).

[0025] f. As used herein, the term “peptide nucleic acid” or “PNA” meansany oligomer, linked polymer or chimeric oligomer, comprising two ormore PNA subunits (residues), including any of the polymers referred toor claimed as peptide nucleic acids in U.S. Pat. Nos. 5,539,082,5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, 5,786,461,5,837,459, 5,891,625, 5,972,610, 5,986,053, 6,107,470 and 6,357,163. Inthe most preferred embodiment, a PNA subunit consists of a naturallyoccurring or non-naturally occurring nucleobase attached to the azanitrogen of the N-[2-(aminoethyl)]glycine backbone through a methylenecarbonyl linkage.

[0026] g. As used herein, the terms “label” and “detectable moiety” areinterchangeable and shall refer to moieties that can be attached to aprobe to thereby render the probe detectable by an instrument or method.

[0027] h. As used herein, the term “locked nucleic acid” or “LNA” meansany oligomer, linked polymer or chimeric oligomer, comprising one ormore LNA subunits (residues), including any of the polymers referred toor claimed as locked nucleic acids, and nucleic acid analogs in U.S.Pat. Nos. 6,639,059, 6,670,461, U.S. patent application Nos.US2003077609 A1, US2003224377 A1, US2003082807 A1 and World PatentOffice Document number WO03095467. In the most preferred embodiment, aLNA subunit consists of a naturally occurring or non-naturally occurringribonucleoside in which the 4′ oxygen is joined to the 2′ carbon througha methylene linkage.

[0028] i. Reference herein to “all species of the genus Pseudomonas”, ora related phrase means essentially all species of that genus describedin the “Approved lists of bacterial names.” Int. J. Syst. Bacteriol.(1980) 30:225-420 with subsequent revisions published in Int. J. Syst.Bacteriol. with the exception of Pseudomonas pertucinogena (see Example2)

[0029] 2. Description

[0030] I. General

[0031] PNA Synthesis:

[0032] Methods for the chemical assembly of PNAs are well known (see:U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336,5,773,571, 5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053 and6,107,470).

[0033] PNA Labeling:

[0034] Preferred non-limiting methods for labeling PNAs are described inU.S. Pat. Nos. 6,110,676, 6,361,942, 6,355,421, the examples section ofthis specification or are otherwise well known in the art of PNAsynthesis and peptide synthesis.

[0035] Labels:

[0036] Non-limiting examples of detectable moieties (labels) suitablefor labeling PNA probes used in the practice of this invention wouldinclude a dextran conjugate, a branched nucleic acid detection system, achromophore, a fluorophore, a spin label, a radioisotope, an enzyme, ahapten, an acridinium ester and a chemiluminescent compound.

[0037] Other suitable labeling reagents and preferred methods ofattachment would be recognized by those of ordinary skill in the art ofPNA, peptide or nucleic acid synthesis.

[0038] Preferred haptens include 5 (6)-carboxyfluorescein,2,4-dinitrophenyl, digoxigenin, and biotin.

[0039] Preferred fluorochromes (fluorophores) include 5(6)-carboxyfluorescein (Flu), 6-((7-amino-4-methylcoumarin-3-acetyl)amino) hexanoic acid (Cou), 5 (and 6)-carboxy-X-rhodamine (Rox), Cyanine2 (Cy2) Dye, Cyanine 3 (Cy3) Dye, Cyanine 3.5 (Cy3.5) Dye, Cyanine 5(Cy5) Dye, Cyanine 5.5 (Cy5.5) Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9)Dye (Cyanine dyes 2,3,3.5,5 and 5.5 are available as NHS esters fromAmersham, Arlington Heights, Ill.), JOE, Tamara or the Alexa dye series(Molecular Probes, Eugene, Oreg.).

[0040] Preferred enzymes include polymerases (e. g. Taq polymerase,Klenow PNA polymerase, T7 DNA polymerase, Sequenase, DNA polymerase 1and phi29 polymerase), alkaline phosphatase (AP), horseradish peroxidase(HRP) and most preferably, soy bean peroxidase (SBP).

[0041] Unlabeled Probes:

[0042] The probes that are used for the practice of this invention neednot be labeled with a detectable moiety to be operable within themethods of this invention, for example when attached to a solid support

[0043] Self-Indicating (or Reporting) Probes:

[0044] Beacon probes are examples of self-indicating probes whichinclude a donor moiety and a acceptor moiety. The donor and acceptormoieties operate such that the acceptor moieties accept energytransferred from the donor moieties or otherwise quench signal from thedonor moiety. Though the previously listed fluorophores (with suitablespectral properties) might also operate as energy transfer acceptors,preferably, the acceptor moiety is a quencher moiety. Preferably, thequencher moiety is a non-fluorescent aromatic or heteroaromatic moiety.The preferred quencher moiety is 4-((4-(dimethylamino) phenyl) azo)benzoic acid (dabcyl). In a preferred embodiment, the self-indicatingBeacon probe is a PNA Linear Beacon as more fully described in U.S. Pat.No. 6,485,901.

[0045] In another embodiment, the self-indicating probes of thisinvention are of the type described in WIPO patent applicationWO97/45539. These self-indicating probes differ as compared with Beaconprobes primarily in that the reporter must interact with the nucleicacid to produce signal.

[0046] Spacer/Linker Moieties:

[0047] Generally, spacers are used to minimize the adverse effects thatbulky labeling reagents might have on hybridization properties ofprobes. Preferred spacer/linker moieties for the nucleobase polymers ofthis invention consist of one or more aminoalkyl carboxylic acids (e. g.aminocaproic acid), the side chain of an amino acid (e. g. the sidechain of lysine or omithine), natural amino acids (e. g. glycine),aminooxyalkylacids (e. g. 8-amino-3,6-dioxaoctanoic acid), alkyl diacids(e. g. succinic acid), alkyloxy diacids (e. g. diglycolic acid) oralkyldiamines (e. g. 1,8-diamino-3,6-dioxaoctane). Preferably, suchlinker moieties will includes less than about 10 subunits, preferablyless than about 8 subunits, with about 1 to about 5 subunits beinguseful for many applications.

[0048] Hybridization Conditions/Stringency:

[0049] Those of ordinary skill in the art of nucleic acid hybridizationwill recognize that factors commonly used to impose or controlstringency of hybridization include formamide concentration (or otherchemical denaturant reagent), salt concentration (i.e., ionic strength),hybridization temperature, detergent concentration, pH and the presenceor absence of chaotropes. Optimal stringency for a probe/target sequencecombination is often found by the well known technique of fixing severalof the aforementioned stringency factors and then determining the effectof varying a single stringency factor. The same stringency factors canbe modulated to thereby control the stringency of hybridization of a PNAto a nucleic acid, except that the hybridization of a PNA is fairlyindependent of ionic strength. Optimal stringency for an assay may beexperimentally determined by examination of each stringency factor untilthe desired degree of discrimination is achieved.

[0050] Suitable Hybridization Conditions:

[0051] Generally, the more closely related the background causingnucleic acid contaminates are to the target sequence, the more carefullystringency must be controlled. Blocking probes may also be used as ameans to improve discrimination beyond the limits possible by mereoptimization of stringency factors; Suitable hybridization conditionswill thus comprise conditions under which the desired degree ofdiscrimination is achieved such that an assay generates an accurate(within the tolerance desired for the assay) and reproducible result.

[0052] Aided by no more than routine experimentation and the disclosureprovided herein, those of skill in the art will easily be able todetermine suitable hybridization conditions for performing assaysutilizing the methods and compositions described herein. Suitablein-situ hybridization or PCR conditions comprise conditions suitable forperforming an in-situ hybridization or PCR procedure. Thus, suitablein-situ hybridization or PCR conditions will become apparent to those ofskill in the art using the disclosure provided herein, with or withoutadditional routine experimentation.

[0053] Blocking Probes:

[0054] Blocking probes are nucleic acid or non-nucleic acid probes thatcan be used to suppress the binding of the probing nucleobase sequenceof the probing polymer to a non-target sequence. Preferred blockingprobes are PNA probes (see: U.S. Pat. No. 6,110,676). It is believedthat blocking probes operate by hybridization to the non-target sequenceto thereby form a more thermodynamically stable complex than is formedby hybridization between the probing nucleobase sequence and thenon-target sequence. Formation of the more stable and preferred complexblocks formation of the less stable non-preferred complex between theprobing nucleobase sequence and the non-target sequence. Thus, blockingprobes can be used with the methods, kits and compositions of thisinvention to suppress the binding of the probes to a non-target sequencethat might be present and interfere with the performance of the assay.

[0055] Blocking probes are particularly advantageous in single basediscrimination.

[0056] Probing Nucleobase Sequence:

[0057] The probing nucleobase sequence of a probe of this invention isthe specific sequence recognition portion of the construct. Therefore,the probing nucleobase sequence is a nucleobase sequence designed tohybridize to a specific target sequence wherein the presence, absence oramount of the target sequence can be used to directly or indirectlydetect the presence, absence or number of organisms of interest in asample. Consequently, with due consideration to the requirements of aprobe for the assay format chosen, the length and sequence compositionof the probing nucleobase sequence of the probe will generally be chosensuch that a stable complex is formed with the target sequence undersuitable hybridization conditions.

[0058] The preferred probing nucleobase sequence of the probes of thisinvention that are suitable for the analysis of Pseudomonas (sensustricto) comprise a nucleobase sequence CCT ACC ACC TTA MC (Seq. IdNo. 1) and the complements thereto.

[0059] This invention contemplates that variations in these identifiedprobing nucleobase sequences shall also provide probes that are suitablefor the detection, identification and/or quantitation of Pseudomonas(sensu stricto). Variation of the probing nucleobase sequences withinthe parameters described herein is considered to be an embodiment ofthis invention.

[0060] Common variations include, deletions, insertions and frameshifts. Additionally, a shorter probing nucleobase sequence can begenerated by truncation of the sequence identified above.

[0061] A probe of this invention will generally have a probingnucleobase sequence that is exactly complementary to the targetsequence. Alternatively, a substantially complementary probingnucleobase sequence might be used since it has been demonstrated thatgreater sequence discrimination can be obtained when utilizing probeswherein there exists one or more point mutations (base mismatch) betweenthe probe and the target sequence (See: Guo et al., Nature Biotechnology15: 331-335 (1997)). Consequently, the probing nucleobase sequence maybe only 90% homologous to the probing nucleobase sequences identifiedabove. Substantially complementary probing nucleobase sequence withinthe parameters described above is considered to be an embodiment of thisinvention.

[0062] Complements of the probing nucleobase sequence are considered tobe an embodiment of this invention, since it is possible to generate asuitable probe if the target sequence to be detected has been amplifiedor copied to thereby generate the complement to the identified targetsequence.

[0063] Detection, Identification and/or Enumeration:

[0064] By detection is meant analysis for the presence or absence of theorganism optionally present in the sample. By identification is meantestablishment of the identity of the organism by genus and species name.By quantitation is meant enumeration of the organisms in a sample. Someassay formats provide simultaneous detection, identification andenumeration (for example see Stender, H. et al., J. Microbiol. Methods.45:31-39 (2001), others provide detection and identification (forexample see Stender, H. et al., Int. J. Tuberc. Lung Dis. 3:830-837(1999) and yet other assay formats just provide identification (forexample see Oliveira, K et al. J. Clin. Microbiol. 40:247-251 (2002)).

[0065] Antibiotic Resistance

[0066] By determination of resistance to antibiotics is meant analysisof an organism susceptibility to antibiotics based on specific genes ormutations associated with resistance or susceptibility to antimicrobialagents.

[0067] As discussed, in one aspect the invention relates to a PNA probethat includes a nucleobase sequence suitable for the detection,identification and/or quantitation of Pseudomonas (sensu stricto) inwhich a preferred embodiment features a PNA probe (or complementthereof) that is complementary to a target sequence of 23S rRNA or rDNAof essentially all species of the genus Pseudomonas. Preferred PNAprobes will have a length that is generally less than about 30 to about35 subunits, preferably less than about 20 subunits with between fromabout 12 to about 18 subunits being preferred for many applications.

[0068] By the phrase “complementary” is meant relatively closerelationship between the sequence of the PNA probe and its intendednucleic acid template sequence. The percent complementarity between aparticular sequence and its template as described in this applicationcan be determined by standard procedures. The degree of complementaritybetween two sequences can be expressed in a variety of formats includingthe percentage of homology or identity.

[0069] For instance, to determine the percent homology of nucleic acidsequences, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first nucleic acidsequence for optimal alignment with a second nucleic acid sequence). Thenucleotides at corresponding positions are then compared. When aposition in the first sequence is occupied by the samenucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent homology between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=# of identical positions/total # ofpositions (e.g., overlapping positions). multiplied by.100). In oneembodiment the two sequences are the same length.

[0070] To determine percent homology between two sequences, thealgorithm of Karlin and Altschul (1990) Proc. Natl. Acad Sci. USA87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90:5873-5877 is used. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol.Biol. 215:403410. BLAST nucleotide searches are performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleobase sequence described herein. To obtain gappedalignments for comparison purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) are used.

[0071] Such manipulations are readily adapted to determine the percenthomology between a PNA probe sequence and its corresponding targetnucleic acid template. Sequences that are completely homologous withrespect to one another are sometimes referred to herein as being“identical”.

[0072] Accordingly, and in one embodiment, at least a portion of one ormore of the foregoing probes is at least about 90% identical to thePseudomonas target sequence, preferably at least about 95% identical,more preferably at least about 98% to 100% identical to that sequence.By “at least a portion” of the probe is meant generally less than about14 subunits, preferably between from about 9 to about 14 subunits suchas about 10 to about 13 subunits.

[0073] A generally preferred PNA probe for many invention applicationsincludes (or in some embodiments essentially consists of) the followingsequence: CCT ACC ACC TTA MC as well as the complement of that sequence.Sometimes the sequence (and its complement) is referred to as a“preferred probing nucleobase” sequence or related phrase.

[0074] Although not usually preferred, the preferred probing nucleobasesequence may include additional PNA, DNA or LNA subunits, for instance,added to an end of the sequence, to both ends of the sequence, and/orbetween the ends (eg., 1, 2, 3, up to about 5, 6, 7 or 8 PNA subunits)in some cases. In such embodiments, the resulting sequence preferablyexhibits good hybridization to the intended Pseudomonas target sequence.That is, hybridization is not substantially impaired when compared tohybridization under the same conditions with the preferred probingnucleobase sequence. Specific binding between a given PNA probe and thetarget sequence can be monitored by a variety of suitable techniquessuch as those described in Stender H et al. PNA for rapid microbiology.J Microbiol Methods. 2002 Jan;48(1):1-17. Such methods further includedetermining the difference in Tm (ΔTm) between the probe and targetsequence and the probe and non-target sequence(s).

[0075] A variety of hybridization conditions have been described indetail in Williams B et al, PNA fluorescent in situ hybridization forrapid microbiology and cytogenetic analysis.

[0076] One or more deletions, substitutions (or both) of the preferredprobing nucleobase sequence are also contemplated (eg., less then about8 subunits, such as about 1, 2, 3, 4, or about 5 subunits), providedhybridization to the intended Pseudomonas target sequence is notsubstantially impaired when compared to the preferred probing nucleobasesequence itself. By the phrase “not substantially impaired” is meantthat the modified nucleobase sequence provides sufficient discriminationbetween target and non-target sequences under suitable hybridizationconditions. By the phrase “sufficient discrimination” is meant that atarget binding complex and a non-target binding complex exhibit a ΔTmgreater than about 2° C., preferably greater than 5° C., most preferablygreater than 10° C. eg, between from about 2° C. to about 75° C. Methodsof determining such ΔTm are known. By the phrase “suitable hybridizationconditions” is meant conditions such as those described by See H.Stender et al., supra. A suitable hybridization condition for performingthe analysis includes, but is not limited to, the conditions describedbelow in the Example.

[0077] Preferred deletions occur at an end of the sequence, at both endsor between such ends. An example of a substitution in accordance withthe invention is T=>U.

[0078] Although usually not necessary, the preferred probing nucleobasesequence can be adapted to include at least one of: 1) a subunit deletedtherefrom, 2) a subunit added thereto and 3) a substituted subunit; forexample, 1, 2, 3, 4, or about 5 of such sequence changes. Such changescan occur at the end of the sequence, at both ends or between such endsas needed. Preferably, hybridization to the intended Pseudomonas targetis not substantially impaired when compared with preferred probingnucleobase sequence. Hybridization can be determined as discussed above.

[0079] A variety of standard procedures exist for monitoring and (ifdesired) quantifying hybridization between two sequences including, butnot limited to, the above-mentioned procedures.

[0080] Collectively, the foregoing changes to the sequence of thepreferred probing nucleobase sequence are sometimes called “variations”or “variants” to indicate change in sequence with respect to thepreferred probing nucleobase sequence (or its complement). Variations ofthe probing nucleobase sequence are thus considered to be an embodimentof this invention. Common variations have already been described andgenerally include, deletions, insertions, substitutions and frameshifts. Additionally, a shorter probing nucleobase sequence can begenerated by truncation of the sequence identified above. Preferredvariations do not substantially impair hybridization when compared tothe preferred nucleobase sequence.

[0081] Further probes of the invention will comprise at least a probingnucleobase sequence (as previously described herein) and at least onedetectable moiety as defined here. Non-limiting examples of additionalmoieties include linkers, spacers, natural or non-natural amino acids,or other subunits of PNA, LNA, DNA or RNA. Still further variations ofthe preferred probing nucleobase sequence include certain nucleobasederivatives such as methylcytosine, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudoisocytosine, 2-thiouracil, 2-thiothymidine uracil and the like.

[0082] Additional probes according to the invention can be labeled withone or a combination of suitable detectable moieties such as one, two orthree of same. Internal labeling of the probe is also contemplated. Avariety of acceptable moieties have been disclosed herein.

[0083] Further probes in accord with the invention are self-indicating(or self-reporting) which probes preferably have a PNA Linear Beaconformat as described herein.

[0084] Additionally suitable PNA probes of the invention are unlabeledand in some instances may be bound covalently or non-covalently to asuitable solid support. Examples of suitable supports have beendisclosed in U.S. Pat. No. 6,664,045, for instance.

[0085] Further probes according to the invention will include at leastone spacer or linker group that is preferably adapted to help space thedetectable moiety from the probing sequence. A variety of suitablespacer/linkers have already been described.

[0086] A preferred use of one or combination of the foregoing PNA probesis in the in situ hybridization analysis of Pseudomonas (sensu stricto)that is optionally present in a sample. By “optionally present” is meantthat the bacteria is known to be in the sample or it is suspected to bein the sample.

[0087] As discussed, the invention features a method for the detection,identification and/or quantitation of Pseudomonas (sensu stricto) in asample. The analysis can be accomplished by nearly any procedureincluding in situ analysis, fluorescence in situ hybridization and thelike. Preferred analytical methods do not rely substantially on use ofcross-linking reagents or enzymes prior to hybridization between theprobing sequence and the intended target. More preferably, the analysisavoids such use entirely and does not involve the use of cross-linkingreagents or enzymes prior to hybridization. Particular probing sequencesfor use with the method include any of the forgoing probes including thepreferred nucleobase sequence and variants thereof.

[0088] More preferred invention methods involve use to detect a nucleicacid that includes a target sequence in which the nucleic acid has beenpreviously manipulated such as by synthesis or amplification usingstandard procedures. Preferred nucleic acid synthesis and amplificationreactions have already been discussed and include at least one ofPolymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), StrandDisplacement Amplification (SDA), Transcription-Mediated Amplification(TMA), Rolling Circle Amplification (RCA) and Q beta replicase, forexample.

[0089] Practice of the invention is flexible as it can accommodate oneor a combination of procedures to improve or otherwise enhance specificbinding between an invention probe and the intended target. Forinstance, and in one embodiment, the method further includes adding atleast one blocking probe to method, preferably to reduce or eliminateany hybridization of the PNA probe to non-target sequence.

[0090] Use of the invention is also flexible in the sense that it can beused in a wide variety of assay formats. For instance, and in oneembodiment, the target sequence is immobilized to a surface. Examples ofsuch surfaces have already been described but generally include suitablepolymer or paper supports, beads, and the like. Alternatively, or inaddition, a probe of the invention is one component of an array.

[0091] Preferred samples for use with the invention are biologicalsamples such as those obtained from blood (including plasma), urine, asecretion, sweat, pus, sputum, stool, mucous or cultures thereof.

[0092] As mentioned, the invention also features a kit that has beenadapted to perform an assay for detection, identification and/orquantitation of Pseudomonas (sensu stricto) in a sample. Typically, sucha kit includes a) at least one of the probe disclosed herein such as thepreferred nucleobase sequence and b) other reagents or compositionsnecessary or helpful to perform the assay (eg., sterile water, buffer,and directions for using the kit and the like). By “adapted” is meantthat the kit includes a kit component useful for detecting, identifyingand/or quantifying Pseudomonas in the sample. Examples of such materialsinclude the preferred nucleobase sequence as well as variants thereof.Other examples include one or more components to perform an assay suchas an in-situ hybridization or real-time PCR assay.

[0093] In one kit embodiment, it will often be helpful to include apositive control such as a sample with a known Pseudomonas species. Inthis invention example, any microorganisms present in the sample can beindependently detected, identified and/or quantitated, preferably byreference to the positive control. It will be appreciated that use ofthe positive control need not accompany every invention application suchas when the properties of particular sample or sample set is well known(eg., clinical samples).

[0094] A kit in accord with the invention has a wide variety ofimportant applications. In one embodiment, the kit is adapted to detect,identify and/or quantitate the amount of any Pseudomonas in a sample inwhich the sample has been exposed to appropriate antimicrobial agents.The invention is thus particularly useful to monitor the effectivenessof new and known antimicrobials.

[0095] Such a kit can be used with one or a combination of detectionformats as described herein including, but not limited to, in-situhybridization assay and a real-time PCR assay. Such kits find particularuse in the examination of clinical, industrial, medical, research andfoodstuff samples including clinical specimens. The kit may be used withcultures made from the samples if needed. Other kit uses include use inthe testing of food, beverages, water, pharmaceutical products, personalcare products, dairy products or environmental samples or culturesthereof.

[0096] There follows a discussion of more preferred inventionembodiments.

II. PREFERRED EMBODIMENTS OF THE INVENTION

[0097] a. PNA Probes:

[0098] In one embodiment, this invention is directed to PNA probes. ThePNA probes of this invention are suitable for detecting, identifyingand/or quantitating Pseudomonas (sensu stricto) optionally present in asample. General characteristics (e.g. length, labels, nucleobasesequences, linkers etc.) of PNA probes suitable for the detection,identification and/or quantitation of Pseudomonas (sensu stricto) havebeen previously described herein. The preferred probing nucleobasesequence of PNA probes of this invention are listed in Table 1. SequenceID Nucleobase sequence Seq. Id. No. 1 CCT ACC ACC TTA AAC

[0099] The PNA probes of this invention may comprise only a probingnucleobase sequence (as previously described herein) or may compriseadditional moieties. Non-limiting examples of additional moietiesinclude detectable moieties (labels), linkers, spacers, natural ornon-natural amino acids, or other subunits of PNA, DNA or RNA.Additional moieties may be functional or non-functional in an assay.Generally however, additional moieties will be selected to be functionalwithin the design of the assay in which the PNA probe is to be used. Thepreferred PNA probes of this invention are labeled with one or moredetectable moieties selected from the group consisting of fluorophores,enzymes and haptens.

[0100] In preferred embodiments, the probes of this invention are usedin in-situ hybridization (ISH) and fluorescence in-situ hybridization(FISH) assays. Excess probe used in an ISH or FISH assay typically mustbe removed so that the detectable moiety of the specifically bound probecan be detected above the background signal that results from stillpresent but unhybridized probe. Generally, the excess probe is washedaway after the sample has been incubated with probe for a period oftime. However, the use of self-reporting PNA probes is a preferredembodiment of this invention, since there is no requirement that excessself-indicating probe be completely removed (washed away) from thesample since it generates little or no detectable background. Inaddition to ISH or FISH assays, self-indicating probes comprising theselected probing nucleobase sequence described herein are particularlyuseful in all kinds of homogeneous assays such as in real-time PCR oruseful with self-indicating devices (e. g. lateral flow assay) orself-indicating arrays.

[0101] b. Methods:

[0102] In another embodiment, this invention is directed to a methodsuitable for detecting, identifying and/or quantitating Pseudomonas(sensu stricto) optionally in a sample. The general and specificcharacteristics of PNA probes suitable for the detection, identificationor quantitation of Pseudomonas (sensu stricto) have been previouslydescribed herein. Preferred probing nucleobase sequences are listed inTable 1.

[0103] The method for detecting, identifying and/or quantitatingPseudomonas (sensu stricto) in a sample comprises contacting the samplewith one or more PNA probes suitable for hybridization to a targetsequence which is unique to all species of the genus Pseudomonas. Inpreferred embodiments, the probe comprises a probing nucleobase sequencewherein at least a portion of the probing nucleobase sequence iscomplementary to a target sequence of 23S rRNA or rDNA of all species ofthe genus Pseudomonas and with at least one nucleobase difference to thecorresponding 23S rRNA or rDNA nucleobase sequences of other bacteriumspecies.

[0104] According to the method, Pseudomonas (sensu stricto) in thesample is then detected, identified and/or quantitated. Detection,identification and/or quantitation of Pseudomonas (sensu stricto) ismade possible by correlating hybridization, under suitable hybridizationconditions or suitable in-situ hybridization conditions, of the probingnucleobase sequence of a PNA probe to the target sequence of all speciesof the genus Pseudomonas sought to be detected with the presence,absence or number of the Pseudomonas (sensu stricto) organisms in thesample. Typically, this correlation is made possible by direct orindirect detection of the probe/target sequence hybrid.

[0105] Fluorescence in situ Hybridization and Real-Time PCR:

[0106] The PNA probes, methods, kits and compositions of this inventionare particularly useful for the rapid probe-based detection,identification and/or quantitation of Pseudomonas (sensu stricto). Inpreferred embodiments, in-situ hybridization or PCR is used as the assayformat for detecting, identifying or quantitating Pseudomonas (sensustricto). Most preferably, fluorescence in-situ hybridization (PNA FISH)or real-time PCR is the assay format. (Reviewed by Stender et al. J.Microbiol. Methods 48:1-17 (2002)).

[0107] Preferably, smears for PNA FISH analysis are not treated withcross-linking agents or enzymes prior to hybridization.

[0108] Exemplary Assay Formats:

[0109] Exemplary methods for performing PNA FISH can be found in:Oliveira et., J. Clin. Microbiol 40:247-251 (2002), Rigby et al., J.Clin. Microbiol. 40:2182-2186 (2002), Stender et al., J. Clin.Microbiol. 37:2760-2765 (1999), Perry-O'Keefe et al., J. Microbiol.Methods 47:281-292 (2001). According to one method, a smear of thesample, such as, but not limited to, a positive blood culture, isprepared on microscope slides and covered with one drop of thefluorescein-labeled PNA probe in hybridization buffer. A coverslip isplaced on the smear to ensure an even coverage, and the slide issubsequently placed on a slide warmer or incubator at 55° C. for 90minutes. Following hybridization, the coverslip is removed by submergingthe slide into a pre-warmed stringent wash solution and the slide iswashed for 30 minutes. The smear is finally mounted with one drop ofmounting fluid, covered with a coverslip and examined by fluorescencemicroscopy.

[0110]Pseudomonas optimally present in a sample which may be analyzedwith the PNA probes contained in the kits of this invention can bedetected, identified and/or quantitated by several instruments, such asbut not limited to the following examples: microscope (for example seeOliveira et al., J. Clin. Microbiol 40:247-251 (2002)), film (forexample see Perry-O'Keefe et al., J. Appl. Microbiol. 90:180-189)(2001), camera and instant film (for example see Stender et al., J.Microbiol. Methods 42:245-253 (2000)), luminometer (for example seeStender et al., J. Microbiol. Methods 46:69-75 (2001), laser scanningdevice (for example see Stender et al., J. Microbiol. Methods 45: 31-39(2001) or flow cytometer (for example see Wordon et al., Appl. Environ.Microbiol. 66:284-289 (2000)). Automated slide scanners and flowcytometers are particularly useful for rapidly quantitating the numberof microorganisms present in a sample of interest.

[0111] Exemplary methods for performing real-time PCR usingself-reporting PNA probes can be found in: Fiandaca et al., Abstract,Nucleic Acid-Based technologies. DNA/RNA/PNA Diagnostics, Washington,DC, May 14-16, 2001, and Perry-O'Keefe et al., Abstract, InternationalConference on Emerging Infectious Diseases, Atlanta, 2002.

[0112] d. Kits:

[0113] In yet another embodiment, this invention is directed to kitssuitable for performing an assay, which detects, identifies and/orquantitates Pseudomonas (sensu stricto) optionally present in a sample.The general and preferred characteristics of PNA probes suitable for thedetection, identification or quantitation of Pseudomonas (sensu stricti)have been previously described herein. Preferred probing nucleobasesequences are listed in Table 1. Furthermore, methods suitable for usingthe PNA probes to detect, identify or quantitate Pseudomonas (sensustricto) in a sample have been previously described herein.

[0114] The kits of this invention comprise one or more PNA probes andother reagents or compositions, which are selected to perform an assayor otherwise simplify the performance of an assay used to detect,identify and/or quantitate Pseudomonas (sensu stricto) in a sample.

[0115] e. Exemplary Applications for Using the Invention:

[0116] The PNA probes, methods and kits of this invention areparticularly useful for the detection, identification and/orquantitation of Pseudomonas (sensu stricto) in clinical samples, food,beverages, water, pharmaceutical products, personal care products, dairyproducts or environmental samples and cultures thereof.

[0117] Having described the preferred embodiments of the invention, itwill now become apparent to one of skill in the art that otherembodiments incorporating the concepts described herein may be used. Itis felt, therefore, that these embodiments should not be limited todisclosed embodiments but rather should be limited only by the spiritand scope of the following claims.

EXAMPLE

[0118] This invention is now illustrated by the following example, whichis not intended to be limiting in any way.

[0119] Reference Strains.

[0120] The study included reference strains from American Type CultureCollection (ATCC), Manassas, Va. representing Pseudomonas species andother non-Pseudomonas species, which primarily comprisedPseudomonas-like species, including species that were previouslyincluded in the Pseudomonas genus. An overnight culture grown at 35-37°C. was prepared from each species by standard methods.

[0121] Preparation of Smears.

[0122] For each smear, one drop of PBS with 1% (v/v) Triton X-100(Aldrich) was placed in a 8-mm diameter well of a teflon-coatedmicroscope slide (Erie Scientific, Portsmouth, N.H.) and mixed gentlywith a small drop of re-suspended culture. The slide was then placed ona 60° C. slide warmer for 20 min at which point the smears were dry.Subsequently, the smears were disinfected by immersion into 96% (v/v)ethanol for 5-10 minutes and air-dried.

[0123] Fluorescence in situ Hybridization (FISH).

[0124] Smears were covered with approximately 50 μL of hybridizationsolution containing 10% (w/v) dextran sulfate (Sigma Chemical Co., St.Louis, Mo.), 10 mM NaCl (J. T. Baker), 30% (v/v) formamide (Sigma), 0.1%(w/v) sodium pyrophosphate (Sigma), 0.2% (w/v) polyvinylpyrrolidone(Sigma), 0.2% (w/v) ficoll (Sigma), 5 mM Na₂EDTA (Sigma), 1% (v/v)Triton X-100 (Aldrich), 50 mM Tris/HCl pH 7.5 and 500 nMfluorescein-labeled PNA probe (Flu-OO-CCTACCACCTTAAAC) targetingPseudomonas (sensu stricto). Coverslips were placed on the smears toensure even coverage with hybridization solution, and the slides weresubsequently placed on a slide warmer with a humidity chamber(Slidemoat, Boeckel, Germany) and incubated for 90 min at 50° C.Following hybridization, the coverslips were removed by submerging theslides into approximately 20 mL/slide pre-warmed 5 mM Tris, pH 10, 15 mMNaCl (J. T. Baker), 0.1% Triton X-100 (Aldrich) in a water bath at 50°C. and washed for 30 min. Each smear was finally mounted using one dropof Mounting Fluid and covered with a coverslip. Microscopic examinationwas conducted using a fluorescence microscope equipped with a FITC/TexasRed dual band filter set. Pseudomonas (sensu stricto) was identified asgreen fluorescent rods.

[0125] The results are listed in the Table 1 below. Species ATCC#Results Acinetobacter calcoaceticus 14987 Negative Aeromonas hydrophila7965 Negative Brevundimonas diminuta 19146 Negative Burkholderia cepacia25416 Negative Comamonas testosteroni 17409 Negative Delftia acidovorans15668 Negative Pseudomonas aeruginosa 9027 Positive Pseudomonasaeruginosa 27853 Positive Pseudomonas alcaligenes 14909 PositivePseudomonas chlororaphis 9446 Positive Pseudomonas fluorescens 17397Positive Pseudomonas fluorescens 13525 Positive Pseudomonas fragi 4973Positive Pseudomonas huttiensis 14670 Positive Pseudomonas luteola 35563Positive Pseudomonas mendocina 25411 Positive Pseudomonas mucidolens4685 Positive Pseudomonas nitroreducens 33634 Positive Pseudomonaspertucinogena 190 Negative Pseudomonas pseudoalcaligenes 12815 PositivePseudomonas putida 12633 Positive Pseudomonas putida 17484 PositivePseudomonas stutzeri 11607 Positive Pseudomonas veronii 700474 PositiveRalstonia pickettii 27511 Negative Sphingomonas paucimobilis 29837Negative Stenotrophomonas maltophilia 13637 Negative

[0126] The results show that PNA probe provides accurate identificationof Pseudomonas species only, whereas other species includingPseudomonas-like species were all negative.

[0127] According to Table 1, Pseudomonas pertucinogena was not detectedby the PNA probe. This species belongs to the Pseudomonas pertucinogenagroup, where the other group member Pseudomonas denitrificans has beenexcluded from the Pseudomonas genus (Rejection of the species namePseudomonas denitrificans (Christensen) Bergey et al. 1923.“Int. J.Syst. Bacteriol. (1982) 32:466). Other studies have shown thatPseudomonas pertucinogena is closely related with Bordetella species andmay therefore not belong in the Pseudomonas genus. Even if it is, it isalso believed that presence of Pseudomonas pertucinogena in a sampleused to practice the invention would be rare at best. Thus, the “falsenegative” shown in Table 1 above is not believed to have anysignificance and should not be construed to limit the invention in anyway.

[0128] The invention has been described in detail with reference topreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of this disclosure, maymake modifications and improvements within the spirit and scope of theinvention.

[0129] Equivalents

[0130] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the invention as defined by the appended claims. Those skilled in theart will be able to ascertain, using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed in the scope of the claims.

[0131] The disclosures of all references mentioned herein areincorporated by reference.

1 1 1 15 DNA Artificial Sequence Description of Artificial SequenceSynthetic PNA probe 1 cctaccacct taaac 15

We claim:
 1. A PNA probe comprising a nucleobase sequence suitable forthe detection, identification and/or quantitation of Pseudomonas (sensustricto), said PNA probe being complementary to a target sequence of 23SrRNA or rDNA of all species of the genus Pseudomonas, or its complement.2. The PNA probe of claim 1, wherein at least a portion of the probe isat least about 90% identical to the nucleobase sequence or complementthereof selected from the following sequence: CCT ACC ACC TTA MC (Seq.Id. No. 1).
 3. The PNA probe of claim 1, wherein the probe sequence is8-17 subunits in length.
 4. The PNA probe of claim 1 for the detection,identification and/or quantification of Pseudomonas (sensu stricto)comprising the following probe sequence: CCT ACC ACC TTA MC (Seq. Id.No. 1), the complement and/or variations thereof.
 5. The PNA probe ofclaim 1, wherein the probe is labeled with at least one detectablemoiety.
 6. The PNA probe of claim 5, wherein the detectable moiety ormoieties are selected from the group consisting of: a conjugate, abranched detection system, a chromophore, a fluorophore, a spin label, aradioisotope, an enzyme, a hapten, an acridinium ester and a luminescentcompound.
 7. The PNA probe of claim 5, wherein the probe isself-reporting.
 8. The PNA probe of claims 7, wherein the probecomprises a PNA Linear Beacon.
 9. The PNA probe of claim 1, wherein theprobe is unlabeled.
 10. The PNA probe of claim 1, wherein the probe isbound to a support.
 11. The PNA probe of claims 1, wherein the probefurther comprises a spacer or a linker.
 12. The PNA probe of claims 1,wherein in situ hybridization is used for analysis of Pseudomonas (sensustricto) optionally present in the sample.
 13. A method for thedetection, identification and/or quantitation of Pseudomonas (sensustricto) in a sample, said method comprising: a) contacting at least oneof the PNA probes of claim 1 to the sample, b) hybridizing the PNA probeto a target sequence of species of the genus Pseudomonas in the sample;and c) detecting the hybridization as being indicative of presence,identity and/or amount of Pseudomonas (sensu stricto) in the sample. 14.A method according to claim 13, wherein the analysis takes place insitu.
 15. A method according to claim 12, wherein the analysis takesplace by fluorescence in situ hybridization.
 16. A method according toclaims 15, wherein the analysis does not involve the use ofcross-linking reagents or enzymes prior to hybridization.
 17. The methodof claim 12, wherein the method is used to detect a nucleic acidcomprising a target sequence wherein said nucleic acid has beensynthesized or amplified in a reaction.
 18. The method of claim 17,wherein preferred nucleic acid synthesis or nucleic acid amplificationreactions are selected from the group consisting of: Polymerase ChainReaction (PCR), Ligase Chain Reaction (LCR), Strand DisplacementAmplification (SDA), Transcription-Mediated Amplification (TMA), RollingCircle Amplification (RCA) and Q beta replicase.
 19. The method of claim12, wherein the method further comprises adding at least one blockingprobe to reduce or eliminate any hybridization of the PNA probe tonon-target sequence.
 20. The method of claim 12, wherein the targetsequence is immobilized to a surface.
 21. The method of claim 12,wherein said PNA probe is immobilized to a surface.
 22. The method ofclaim 21, wherein said PNA probe is one component of an array.
 23. Themethod of claim 12, wherein the sample is a biological sample.
 24. Themethod of claim 23, wherein the biological sample is blood, urine,secretion, sweat, sputum, stool, mucous, or cultures thereof.
 25. A kitadapted to perform an assay for the detection, identification and/orquantitation of Pseudomonas (sensu stricto) in a sample, wherein saidkit comprises: a) a PNA probe according to claim 1 and b) other reagentsor compositions necessary to perform the assay.
 26. The kit of claim 25,wherein Pseudomonas (sensu stricto) and at least one other microorganismoptionally present in a sample are independently detected, identifiedand/or quantitated.
 27. The kit of claim 25, wherein Pseudomonas (sensustricto) optionally present in a sample is detected, identified and/orquantitated and its susceptibility to antimicrobial agents isdetermined.
 28. The kit of claim 25, wherein the kit is further adaptedto perform in an in-situ hybridization assay.
 29. The kit of claim 25,wherein the kit is further apdapted to perform a real-time PCR assay.30. The kit of claim 25, wherein the kit is adapted to examine clinicalsamples such as clinical specimens or cultures thereof.
 31. The kit ofclaim 25, wherein the kit is adapted to examine food, beverages, water,pharmaceutical products, personal care products, dairy products orenvironmental samples or cultures thereof.